5456–5467 Nucleic Acids Research, 2014, Vol. 42, No. 9
doi: 10.1093/nar/gku173
Published online 05 March 2014
Genome-wide analysis of regulation of gene
expression and H3K9me2 distribution by JIL-1 kinase
mediated histone H3S10 phosphorylation in
Drosophila
Weili Cai1 , Chao Wang1 , Yeran Li1 , Changfu Yao1 , Lu Shen1 , Sanzhen Liu2 , Xiaomin Bao1 ,
Patrick S. Schnable2,3 , Jack Girton1 , Jørgen Johansen1 and Kristen M. Johansen1,*
1
2
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA,
Department of Agronomy, Iowa State University, Ames, IA 50011, USA and 3 Data2Bio LLC, Ames, IA 50011, USA
Received November 9, 2012; Revised February 09, 2014; Accepted February 11, 2014
In this study we have determined the genome-wide
relationship of JIL-1 kinase mediated H3S10 phosphorylation with gene expression and the distribution of the epigenetic H3K9me2 mark. We show in
wild-type salivary gland cells that the H3S10ph mark
is predominantly enriched at active genes whereas
the H3K9me2 mark is largely associated with inactive genes. Comparison of global transcription profiles in salivary glands from wild-type and JIL-1 null
mutant larvae revealed that the expression levels of
1539 genes changed at least 2-fold in the mutant and
that a substantial number (49%) of these genes were
upregulated whereas 51% were downregulated. Furthermore, the results showed that downregulation of
genes in the mutant was correlated with higher levels
or acquisition of the H3K9me2 mark whereas upregulation of a gene was correlated with loss of or diminished H3K9 dimethylation. These results are compatible with a model where gene expression levels are
modulated by the levels of the H3K9me2 mark independent of the state of the H3S10ph mark, which is
not required for either transcription or gene activation to occur. Rather, H3S10 phosphorylation functions to indirectly maintain active transcription by
counteracting H3K9 dimethylation and gene silencing.
INTRODUCTION
The JIL-1 kinase localizes specifically to euchromatic interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at in* To
terphase in Drosophila (1,2). Genetic interaction assays with
JIL-1 hypomorphic and null allelic combinations demonstrated that JIL-1 can counterbalance the gene-silencing effect of the three major heterochromatin markers H3K9me2,
Su(var)3-7 and HP1a on position-effect variegation and
that in the absence of histone H3S10 phosphorylation these
epigenetic marks spread to ectopic locations on the arms of
polytene chromosomes (3–7). These observations suggested
a model for a dynamic balance between euchromatin and
heterochromatin (3,5,6,8), where the level of gene expression is determined by antagonistic functions of the euchromatic H3S10ph mark on the heterochromatic H3K9me2
mark. In strong support of this model, Wang et al. (6,9) recently provided evidence that H3K9me2 levels at reporter
genes inversely correlate with their levels of expression and
that H3K9me2 levels in turn are regulated by H3S10 phosphorylation. Thus, taken together these findings suggest
that a major function of JIL-1-mediated histone H3S10
phosphorylation is to maintain an active state of chromatin
by counteracting H3K9 dimethylation and gene silencing
(3,6,9,10). In an alternative scenario, Corces et al. have proposed that JIL-1 and histone H3S10 phosphorylation are
required for active transcription by the RNA polymerase
II machinery (11–13). However, the results of these studies
have been controversial because it has been demonstrated
that RNA polymerase II mediated transcription occurs at
robust levels in the absence of H3S10 phosphorylation in
Drosophila (10,14,15).
In this study, to explore the global interplay between the
epigenetic H3S10ph and H3K9me2 chromatin modifications and gene expression, we conducted a genome-wide
analysis of their enriched sites and combined it with an analysis of changes to the distribution of the H3K9me2 mark
and of whole genome transcription level changes in the absence of H3S10 phosphorylation. In order to have the abil-
whom correspondence should be addressed. Tel: +515 294 7959; Fax: 515 294 4858; Email: kristen@iastate.edu
Correspondence may also be addressed to Jørgen Johansen. Tel: +515 294 2358; Email: jorgen@iastate.edu
C The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Downloaded from http://nar.oxfordjournals.org/ at Iowa State University on May 27, 2014
ABSTRACT
Nucleic Acids Research, 2014, Vol. 42, No. 9 5457
MATERIALS AND METHODS
Drosophila melanogaster stocks
Fly stocks were maintained according to standard protocols
(16). Canton S. was used for wild-type preparations. The
JIL-1z2 allele is described in Wang et al. (2) and in Zhang
et al. (17).
ChIP-sequencing and data analysis
For ChIP-sequencing, 50 pairs of salivary glands per sample were dissected from third instar larvae and fixed for
15 min at room temperature in 1 ml of fixative (50-mM
HEPES at pH 7.6, 100-mM NaCl, 0.1-mM EDTA at pH 8,
0.5-mM EGTA at pH 8, 2% formaldehyde). Preparation of
chromatin for immunoprecipitation was performed as previously described (18). Mouse anti-JIL-1 mAb 5C9 (19),
mouse anti-H3K9me2 mAb 1220 (Abcam), or rabbit antiH3S10ph pAb (Cell Signaling or Active Motif) were used
for immunoprecipitation. For each sample, 10% of the chromatin lysate was used as control input DNA, and 90% was
immunoprecipitated with the antibody. DNA from the immunoprecipitated chromatin fragments was purified using
a Wizard SV DNA purification kit (Promega).
Before sequencing, the purified ChIP-enriched DNA
fragments were selected on an agarose gel for fragments 200
bp in size, linkers were added, and the library was amplified by polymerase chain reaction (PCR). Each library was
sequenced at the Iowa State University DNA facility by a
Genome Analyser II or a Hiseq200 sequencing platform
(Illumina). All samples were processed using 36 or 100 bp
single-end protocols (Illumina). All reads were mapped to
dm3 version 5 of the Drosophila melanogaster genome using Bowtie V0.12.7 (20) with the default settings and out-
put to the SAM format. SAMtools V0.1.18 (21) and BEDtools V2.14.3 (22) were used to sort and transfer files. Enriched islands for JIL-1, H3S10ph and H3K9me2 (wildtype and JIL-1Z2 / JIL-1Z2 ) were identified using SICER
V1.1 with 200 bp window size, 200 bp gap size, and an effective genome size of 72% of the Drosophila genome (23).
Enriched islands with a false discovery rate (FDR) of 1%
or less were considered to be valid. MACS V1.4 (24) was
used to identify JIL-1 binding peaks with default settings
and FDRs of 10% or less. Binding islands or the number
of reads obtained in each genomic region scaled to the total number of reads were rendered in the integrated genome
viewer (IGV 2.0). ChIP on chip profiles of Chromator were
generated from data in Kc cells obtained by ModENCODE
(25) and presented as average log2 signal ratio of IP over input. The ChIPpeakAnno bioconductor R package (26) was
employed to annotate the binding sites to the nearest start
of a gene and to map the distance to the nearest TSS. Density plots of the center of enriched binding islands relative to
the distance from the TSS of nearby genes were made using
SAS (SAS Institute, Inc).
RNA-sequencing and data analysis
For RNA-sequencing, total RNA from third instar salivary
glands was isolated using the UltraClean Tissue and Cells
RNA Isolation Kit (Mo Bio). DNA was removed using the
DNase I kit (Mo Bio). Two replicated samples of each wildtype and JIL-1z2 /JIL-1z2 null mutant RNA were amplified
and sequenced on an Illumina Hiseq2000 at the Iowa State
University DNA facility using a paired-end protocol. Lowquality nucleotides were removed from raw reads using
Data2Bio’s trimming script (27). GSNAP (Genomic Shortread Nucleotide Alignment Program, version 201310–12)
(28), which allows for intron-spanning alignments, was used
to map trimmed reads to the reference genome (BDGP
5.72/dm3 June 2013). Reads with one unique best match in
the reference genome (BDGP 5.72/dm3 June 2013) with ≤2
mismatches every 50 bp were used for all subsequent analyses. The read depth of each gene was computed based on the
coordinates of mapped reads and the annotated locations
of genes in the reference genome (BDGP 5.72/dm3 June
2013). Reads per kilobase of exons per million uniquely
mapped reads (RPKM) values were calculated as in Mortazavi et al. (29).
For identification of genes with different expression in
the JIL-1 mutant background, the log-scale 75th percentile
of library size normalization method was used to normalize wild-type and mutant RNA-Seq reads (30). The normalized reads were counted by HTseq(v0.5.4). The QuasiSeq (R package) was used to identify differentially expressed genes by negative binomial quasi-likelihood estimation (31). Genes were considered differentially expressed between wild-type and the mutant if they exhibited a greater
than 2-fold change in expression and an FDR smaller
than 0.05. Scatter plot representations of gene expression
changes between wild-type and JIL-1 null mutant salivary
gland cells as well as regression analysis were performed using SAS (SAS Institute, Inc). Gene Ontology (GO) term categories for genes identified as down- or upregulated in JIL1 null mutant versus wild-type salivary glands were iden-
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ity to specifically map and correlate the location of JIL1 and H3K9me2 with the locations of the histone H3S10
phosphorylation mark, salivary gland cells from third instar
larvae were analyzed. Salivary gland nuclei are all at interphase excluding contributions from mitotic histone H3S10
phosphorylation. We found that most of the identified JIL1 binding peaks located at or near transcription start sites
(TSS) whereas peaks for both H3S10ph and H3K9me2 enrichment were located around 600 bp downstream of the
TSS. A comparison of the transcriptome profiles of salivary glands from wild-type and JIL-1 null mutants revealed
that the expression levels of 1539 genes changed at least
2-fold in the mutant. Interestingly, out of these genes the
expression of 66% of normally active genes was repressed,
whereas the expression of most normally inactive genes
(77%) was activated. Furthermore, we show that in the absence of H3S10 phosphorylation the H3K9me2 mark redistributes and becomes upregulated on ectopic sites on the
chromosome arms, especially on the X-chromosome, and
that this H3K9me2 redistribution correlates with the activation of silent genes and the repression of active genes.
Taken together, these results provide direct support for the
model that H3S10 phosphorylation mainly facilitates gene
expression of active genes by maintaining an open chromatin structure at promoter regions by counteracting H3K9
dimethylation.
5458 Nucleic Acids Research, 2014, Vol. 42, No. 9
tified by DAVID (32) in Level 5 of biological process and
molecular function. In addition, box plots, bar charts and
pie charts were made used using the ggplot2 R package (33)
and statistical analysis was performed with R (v3.0.1).
Chromatin immunoprecipitation and qPCR
Immunohistochemistry
Polytene chromosome squash preparations were performed
as in Cai et al. (35) using a 5 min fixation protocol
and double labeled with JIL-1 mAb 5C9 (19) and rabbit anti-histone H3K9me2 (Millipore). DNA was counterstained by Hoechst 33258 (Molecular Probes) in phosphate buffered saline. The appropriate species- and isotypespecific TRITC-, and FITC-conjugated secondary antibodies (Cappel/ICN, Southern Biotech) were used (1:200 dilution). The final preparations were mounted in 90% glycerol
containing 0.5% n-propyl gallate. The preparations were examined using epifluorescence optics on a Zeiss Axioskop
microscope and images were captured and digitized using a
cooled Spot CCD camera. Images were imported into Photoshop where they were pseudocolored, image processed
and merged. In some images non-linear adjustments were
made to the channel with Hoechst labeling for optimal visualization of chromosomes.
Modeling of JIL-1
The structure of JIL-1 was modeled with the I-TASSER
protein prediction server (36,37) and compared to the
RESULTS
Mapping the genome-wide distribution of JIL-1, H3S10ph
and H3K9me2 in salivary gland cells
All DNA and RNA samples analyzed were from postmitotic third instar larval salivary glands that included both
sexes and issues of JIL-1’s and H3S10 phosphorylation’s
role in dosage compensation of the X-chromosome were
not addressed in this study. For comparisons of gene transcription and H3K9me2 distribution in the absence of JIL1 and H3S10 phosphorylation with wild-type we analyzed
DNA and RNA from salivary glands of JIL-1z2 /JIL-1z2 homozygous larvae. JIL-1z2 is a true null allele generated by Pelement mobilization (2). The global transcription profiles
were generated by analysis of two independent RNA-Seq
experiments. Based on the formula of Mortazavi et al. (29)
we calculated that in salivary glands one read per kilobase
of exons per million uniquely mapped reads (RPKM) corresponded to approximately one transcript per cell. Thus,
genes with an RPKM of one or more were considered active, otherwise they were categorized as inactive. We further
classified the active genes into three separate groups with
low, moderate or high expression levels reflected by RPKMs of from 1–5, >5–30, or >30, respectively. For ChIPSeq analysis of JIL-1 genomic binding sites we used a previously characterized and highly specific mAb 5C9 (19) and
conducted two independent experiments. Enriched chromatin binding sites were identified using the SICER algorithm (23) (Figure 1A). The resulting two data sets were
highly correlated (Supplementary Figure S1) and we identified 2819 genes associated with binding sites enriched for
JIL-1 present in both data sets that were used for the following analysis. For ChIP-Seq analysis of the genome-wide
distribution of the H3S10ph mark we used two previously
validated antibodies (6,9) from Cell Signaling (CS) and Active Motif (AM), respectively. As illustrated in Supplementary Figure S1, the two data sets for these antibodies were
strongly correlated and we identified 2948 genes enriched
for H3S10ph present in both data sets that were used for the
subsequent analysis. For analysis of the H3K9me2 mark we
used the mAb 1220 (Abcam) verified for ChIP analysis by
ModENCODE (25) as well as by Wang et al. (6,9) and determined its global distribution in both wild-type and in the
JIL-1 null mutant background. Based on two independent
ChIP-Seq experiments for each condition, we identified 833
genes enriched for H3K9me2 in both data sets for wild-type
and 488 genes enriched for H3K9me2 in both data sets from
the JIL-1 null background (Supplementary Figure S1). To
further validate our RNA-Seq data we confirmed selected
genes with qPCR assays (Supplementary Figure S2).
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ChIP experiments were performed as previously described
(6,18). In short, for ChIP experiments, 50 pairs of salivary
glands per sample were dissected from third instar larvae
and fixed for 15 min at room temperature in 1 ml of fixative (50-mM HEPES at pH 7.6, 100-mM NaCl, 0.1-mM
EDTA at pH 8, 0.5-mM EGTA at pH 8, 2% formaldehyde).
Mouse anti-JIL-1 mAb 5C9 (19), anti-H3K9me2 (Abcam),
anti-GST mAb 8C7 (34), anti-H3S10ph (Cell Signaling, Active Motif) or purified rabbit IgG (Sigma) were used for immunoprecipitation. For each sample, the chromatin lysate
was divided into equal amounts and immunoprecipitated
with experimental and control antibody, respectively. DNA
from the immunoprecipitated chromatin fragments (average length, 500 bp) was purified using a Wizard SV DNA
purification kit (Promega).
R II
Quantitative PCR was carried out using Brilliant
SYBR Green QPCR Master Mix (Stratagene) in conjunction with an Mx4000 (Stratagene) PCR machine. The
primers used for the various qPCR experiments are listed in
Supplementary Table S1. Cycling parameters were 10 min at
95◦ C, followed by 40 cycles of 30 s at 95◦ C, 30 s at 55◦ C and
30 s at 72◦ C. Fluorescence intensities were plotted against
the number of cycles using an algorithm provided by Stratagene. Template levels were quantified using a calibration
curve based on dilution of concentrated DNA. For each
experimental condition, the relative enrichment was normalized to the corresponding control immunoprecipitation
from the same chromatin lysate.
crystal structure of a nucleosome (PDB ID: 1AOI). ITASSER generates models of proteins by excising continuous fragments from Local Meta-Threading-Server
multiple-threading alignments and then reassembling them
using replica-exchange Monte Carlo simulations (38). The
comparison and visualization between the model of JIL1 and the nucleosome was processed and rendered by PyMOL.
Nucleic Acids Research, 2014, Vol. 42, No. 9 5459
Genome-wide DNA-enrichment profiles for JIL-1, H3S10ph
and H3K9me2
In order to determine the relative distribution of JIL-1
and the H3S10ph and H3K9me2 marks in salivary gland
cells, we generated high-resolution genome-wide DNAenrichment profiles as exemplified in Figure 1A and Supplementary Figure S3. In addition, we compared these profiles to that of Chromator obtained by ChIP on chip analysis by ModENCODE in Kc cells (25). A caveat to this
analysis is that the data are from different cells and tissues.
Chromator is a chromodomain-containing protein (34,39)
that is a known binding partner of JIL-1 that co-localizes
with JIL-1 at interband regions in immunolabeled polytene
salivary gland squash preparations (40). To further characterize the enrichment in and around genes, we plotted the
distribution of the middle position of the enriched islands
(clusters of enriched windows) determined by SICER (23)
relative to TSS of genes (Figure 1B). As illustrated in Figure 1B, genome-wide distance correlation revealed that the
majority of the identified JIL-1 enriched islands were centered in close proximity to the TSS within ±500 bp and
that the peak was aligned near 0 bp. Interestingly, the distribution of Chromator-enriched islands with respect to the
TSS was nearly identical to that of JIL-1 (Figure 1B). Furthermore, analysis of discrete binding peaks of JIL-1 determined by the MACS peak-finding algorithm (24) revealed
that they were highly correlated with Chromator binding
peaks and located preferentially to the 5 -end of genes (Figure 1C). These data suggest that JIL-1 and Chromator share
the same specific binding sites although it has been demon-
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Figure 1. High-resolution profiling of JIL-1, H3S10ph and H3K9me2 enriched genomic regions in Drosophila salivary gland cells. (A) Comparison of
enriched genomic islands for Chromator (data from ModENCODE), JIL-1 (data from JIL-1(data set 2)), H3S10ph (data from H3S10ph(CS antibody))
and H3K9me2 on a section of the X chromosome. The location of genes (in red) is indicated below. The enhanced genomic islands were identified using
the SICER algorithm (23) with a 200 bp window and a 200 bp gap size. (B) Density of Chromator, JIL-1, H3S10ph and H3K9me2 enriched islands as
determined in (A) plotted relative to the distance from the TSS of nearby genes. Chromator and JIL-1 on average tend to localize to enriched islands with
a peak centered near the TSS. H3S10ph and H3K9me2 on average tend to localize with a peak about 600 bp after the TSS. (C) JIL-1 enriched binding
peaks strongly correlate with those of Chromator and are preferentially located at the 5 -end of genes. Lane 1: ChIP on chip profile of Chromator (data
from ModENCODE). The data are represented as average log2 signal ratio of immunoprecipitation (IP) over input. Lane 2: ChIP-Seq profile of JIL-1
(data from JIL-1(data set 2)). The height of the peaks represents the number of reads obtained in each region scaled to the total number of reads. Lane
3: ChIP-Seq input profile. The height of the peaks represents the number of reads obtained in each region scaled to the total number of reads. Lane 4:
peak JIL-1 enriched binding regions determined by the MACS peak finding algorithm (24). Lane 5: the location of genes in the depicted section of the X
chromosome.
5460 Nucleic Acids Research, 2014, Vol. 42, No. 9
H3S10 phosphorylation is enriched at active genes and correlated with enhanced gene expression
JIL-1-mediated H3S10 phosphorylation has been proposed
to maintain an active state of chromatin (43). Consistent
with this hypothesis we found that enrichment of JIL-1 and
the H3S10ph mark predominantly were associated with active genes (79% and 67%, respectively) (Figure 2A). In contrast, enrichment of the H3K9me2 mark had a more pronounced association with inactive genes (47%) (Figure 2A).
However, it should be noted that JIL-1 and the H3S10ph
mark were also enriched at many inactive genes (21% and
33%, respectively) whereas the enrichment of the H3K9me2
mark in many cases was associated with active genes (53%)
(Figure 2A). Thus, to further explore the correlation of gene
expression’s dependence on the presence of the H3S10ph
and H3K9me2 marks we determined the average gene expression of genes enriched for the H3S10ph or H3K9me2
mark only and genes enriched for both marks. As illustrated
in Figure 2B, the median gene expression for these groups
of genes was significantly different from each other with
the highest expression of genes enriched for the H3S10ph
mark only and lowest expression of genes enriched with the
H3K9me2 mark only, whereas gene expression for genes enriched for both marks was intermediate. We further compared the relative proportions of high expression, moderate expression, low expression and inactive genes associated
with enriched levels of the H3S10ph or H3K9me2 mark
only and genes enriched for both marks (Figure 2C). The
results show that the proportion of high, moderate and low
expression genes was greater when associated with enriched
levels of the H3S10ph mark only and conversely that the
proportion of inactive genes increased with enriched levels of the H3K9me2 mark only. The relative proportion of
high, moderate and low expressing genes was intermediate
between genes with enrichment for both marks compared to
the distribution of these classes of genes with single marks
(Figure 2C). Thus, taken together these results suggest that
at a genome-wide level the enrichment for the H3S10ph
mark is directly correlated with enhanced gene expression.
Loss of JIL-1 and H3S10 phosphorylation lead to both gene
up- and downregulation
In order to determine the changes in gene transcription
in the absence of H3S10 phosphorylation, we compared
global transcription profiles in salivary glands in two biological replicates from wild-type and JIL-1z2 /JIL-1z2 homozygous null larvae, respectively. The replicate determinations were highly correlated with Pearson’s coefficients (R2 )
of 0.977 for the two wild-type samples and 0.998 for the
two samples from JIL-1 null larvae. The results showed that
out of nearly 15,000 genes analyzed, the expression levels of
1539 genes changed at least 2-fold and with an FDR smaller
than 0.05. Interestingly, 51% of these genes were downregulated whereas 49% showed increased expression levels in
the JIL-1 null mutant background. This is illustrated in
the scatterplot in Figure 3A where each dot represents the
changes in the expression level of each individual gene and
in the box plot in Figure 3B. We further plotted the changes
in gene expression levels of genes that were classified as active or inactive in wild-type salivary glands. As shown in
the box plot in Figure 3C, there was a significant difference
in the expression changes between the two groups of genes.
Most inactive genes had increased expression in the mutant
background whereas active genes on average had a modest
decrease. However, when active genes were further divided
into groups with low, moderate and high expression, there
was a clear trend that the higher the wild-type expression
levels of a gene the more its expression levels were decreased
in the mutant background (Figure 3D). These data suggest
in general that active genes become repressed but that many
hitherto inactive genes become activated in the absence of
H3S10 phosphorylation.
We next analyzed the up- and downregulated genes in
terms of their GO categories (Figure 3E). Strikingly, the
results indicate that the downregulated set of genes in the
mutant is enriched in categories related to salivary gland
function and development whereas the upregulated list is
enriched in DNA repair, cellular response to stress, and
RNA processing and metabolism categories. That genes in
salivary gland and tissue development specific pathways
were particularly affected by downregulation in the mutant
suggests that H3S10 phosphorylation may play a key role
in keeping tissue and developmentally stage-specific genes
transcriptionally active.
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strated that neither depends on the other for its binding (40).
Although the majority of JIL-1 binding peaks map in the
proximity of the TSS, we also found the presence of many
binding regions at the level of exons, introns and the 3 -end
of genes (Figure 1A).
Although H3S10ph enrichment was highly correlated
with that of JIL-1 (Figure 1A and Supplementary Figure
S1), the profile of H3S10ph was markedly different from
that of JIL-1 around the TSS (Figure 1B). H3S10ph’s distribution was much broader and the peak enrichment was
shifted to about 600 bp downstream of the TSS. Furthermore, only 35% of JIL-1-enriched genes were concomitantly
enriched for the H3S10ph mark. This suggests that JIL-1
may not phosphorylate histone H3 at the nucleosome to
which it is bound. To explore this possibility, we modeled
the 3D structure of JIL-1 using the I-TASSER structure
prediction program (37) and compared it to the nucleosome
crystal structure (41). As illustrated in Supplementary Figure S4, considering that JIL-1 is known to bind the tail of
H3 by the end of its carboxy-terminal domain (42) and that
its folded structure is larger than a nucleosome, it is likely
to have the capacity to phosphorylate histone H3 of one
or more nucleosomes some distance away from its actual
binding site depending on the state of higher order nucleosome packaging. Thus, as indicated by the present data, the
distribution of JIL-1 and H3S10 phosphorylation may not
necessarily be coincident. Interestingly, the distribution of
H3K9me2 binding around the TSS closely mirrored that of
H3S10ph (Figure 1B), indicating that these two epigenetic
modifications may occur in similar genomic contexts.
Nucleic Acids Research, 2014, Vol. 42, No. 9 5461
H3K9me2 redistribution in the absence of H3S10 phosphorylation correlates with activation of silent genes and repression
of active genes
One of the consequences of the absence of H3S10 phosphorylation is a redistribution of the H3K9me2 mark from
pericentric heterochromatin to the euchromatic regions of
the chromosome arms (3–7). This is illustrated by the
H3K9me2 immunolabeling of polytene squash preparations from wild-type and JIL-1 null mutant salivary glands
in Figure 4A with the redistribution especially prominent
on the X-chromosome of both males and females (3). Thus,
to further investigate this redistribution on a genome-wide
level we generated maps of H3K9me2 binding by plotting
normalized read numbers in 200 bp bins across the genome
from wild-type and JIL-1 null salivary glands. Figure 4B
shows an example of such maps comparing wild-type and
JIL-1 mutant X-chromosomes. The results show that in
the JIL-1 null mutant background the levels of H3K9me2
on the chromosome arms were markedly increased and
that enriched levels occurred at sites previously not occupied by significant levels of the H3K9me2 mark. Interestingly, although the H3K9me2 mark was enriched on the Xchromosome compared to the autosomes, overall gene expression levels were not statistically different (Figure 4C).
The study of Wang et al. (9) of gene expression of the
white locus provided evidence that H3K9me2 levels at the
white gene directly correlated with its level of expression and
that the H3K9me2 levels in turn were regulated by H3S10
phosphorylation. Based on these findings we reasoned that
in the absence of H3S10 phosphorylation gene repression
should correlate with increased H3K9me2 binding to the
gene, whereas gene activation should be correlated with a
loss or decreased H3K9me2 binding. To explore this hypothesis, we plotted the expression of all moderate and high
expression genes in our data set without enriched levels of
H3K9me2 that acquired the H3K9me2 mark in the JIL-1
mutant. As illustrated by the box plots in Figure 5A, for
94 such genes there was a significant decrease in their aver-
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Figure 2. Expression levels of genes enriched for JIL-1, H3S10ph and/or H3K9me2. (A) Pie charts of the relative proportion of inactive and active
(RPKM≥1) genes enriched for JIL-1, H3S10ph, or H3K9me2, respectively. (B) Comparison of the expression of genes enriched for the H3K9me2 or
H3S10ph mark only and genes enriched for both marks. Gene expression is represented as log2(RPKM + 0.5). The box plot representation in this and
subsequent figures defines 25th to 75th percentiles (boxes), 50th percentile (lines in boxes) and ranges (whiskers, 1.5 times the interquartile range extended
from both ends of the box or the maximal/minimal value). Outliers were removed from the analysis. The distribution of gene expression for all three
categories was significantly different from each other (P-values < 0.005; Pairwise Wilcoxon Rank Sum Tests). (C) Stack bar charts showing the distribution
of expression of genes enriched for the H3K9me2 or H3S10ph mark only and genes enriched for both marks. Genes with an RPKM of less than one were
categorized as inactive whereas active genes were separated into three groups with low, moderate, or high expression levels reflected by RPKMs of from
1–5, >5–30, or >30.
5462 Nucleic Acids Research, 2014, Vol. 42, No. 9
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Figure 3. Gene expression can be either down- or upregulated in the absence of H3S10 phosphorylation. (A) Scatter plot of gene expression changes between wild-type and JIL-1 null mutant salivary gland cells. Negative log10 (P-values) from Fisher’s exact test were plotted against the log2 (mutant/wild-type)
fold change for each gene. Each dot represents a separate gene. Gray lines indicate the cutoffs for 2-fold changes with a FDR equal to 0.05. Downregulated
genes in the JIL-1 mutant are highlighted in light blue and upregulated genes in orange. (B) Box plot of gene expression changes between wild-type and
JIL-1 null mutant salivary gland cells of all genes changed at least 2-fold represented as log2(mutant/wild-type) fold change for each gene. The overall
changes in gene expression between the mutant and wild-type were not statistically significant (P-value = 0.3; Wilcoxon Signed Rank Test). (C) Box plot
of gene expression changes between wild-type and JIL-1 null mutant salivary gland cells of inactive versus active (RPKM ≥ 1) genes that changed at least
2-fold represented as log2(mutant/wild-type) fold change for each gene. The distribution of gene expression for the two categories was significantly different
from each other (P-value < 0.0001; Wilcoxon Rank Sum Test). (D) Box plot of gene expression changes between wild-type and JIL-1 null mutant salivary
gland cells of active genes separated into three groups with low, moderate, or high expression levels (RPKMs of from 1–5, >5–30, or >30, respectively)
that changed at least 2-fold represented as log2(mutant/wild-type) fold change for each gene. The distribution of gene expression for all three categories
was significantly different from each other (low to moderate: P-value < 0.01; moderate to high: P-value < 0.05; low to high: P-value < 0.0001; Pairwise
Wilcoxon Rank Sum Tests). (E) Gene Ontology (GO) term of JIL-1 enriched genes identified as downregulated (left) or upregulated (right) in JIL-1 null
mutant versus wild-type salivary glands from third instar larvae. The number of genes in each category is shown within the bars. The GO term enrichment
was identified by DAVID (32) in Level 5 of biological process and molecular function.
Nucleic Acids Research, 2014, Vol. 42, No. 9 5463
age levels of expression. To further validate these findings,
we performed ChIP assays as in Wang et al. (6,9) for five
randomly selected genes among the genes analyzed above.
Chromatin was immunoprecipitated (ip) from wild-type or
JIL-1 null mutant larval salivary glands using rabbit antiH3S10ph antibody or purified rabbit IgG antibody (negative control) or mAbs to H3K9me2 or GST (negative control). Primers specific for each of the selected genes were
used to amplify the precipitated material. Experiments were
done in triplicate and relative enrichment of DNA from the
H3S10ph and H3K9me2 ips were normalized to the corresponding control antibody ips performed in tandem for
each experimental sample. As illustrated in Figure 5B, at all
five genes the relative enrichment of H3S10ph in the mutant was reduced to background levels whereas there was a
significant increase of from 2- to 7-fold of the enrichment
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Figure 4. H3K9me2 is redistributed and upregulated on the X-chomosome in JIL-1 null mutants. (A) Wild-type (WT) and JIL-1 mutant (JIL-1z2 /JIL-1z2 )
polytene squash preparations labeled with antibody to JIL-1 (in green), H3K9me2 (in red) and with Hoechst (DNA, in blue/gray). The X-chromosome in
the JIL-1 mutant is indicated with an ‘X.’ (B) Comparison of H3K9me2 distribution in wild-type and JIL-1 null mutants. The maps show normalized (linear
normalization by total library size) read numbers (log2(H3K9me2/input)) in 200 bp bins across the X-chromosome scaled from −1 (dark red) to +1 (dark
blue). (C) Box plot of gene expression changes between wild-type and JIL-1 null mutant salivary gland cells of autosomal versus X-chromosomal genes
that changed at least 2-fold represented as log2(mutant/wild-type) fold change for each gene. The distribution of gene expression for the two categories
was similar (P-value = 0.4; Wilcoxon Rank Sum Test).
5464 Nucleic Acids Research, 2014, Vol. 42, No. 9
of the H3K9me2 mark. We next plotted the expression of
all inactive and low expression genes in our data set that
lost the H3K9me2 mark in the JIL-1 mutant. The box plots
in Figure 6A show that for 69 such genes there was a significant increase in their average levels of expression. As
above we validated these findings by performing ChIP assays for five randomly selected genes among the genes analyzed. The results show that at all five genes there was a significant decrease of from 2- to 6-fold of the enrichment of
the H3K9me2 mark (Figure 6B). Furthermore, the results
confirm that in this class of inactive genes there were very
low wild-type levels of H3S10ph. Taken together, these findings suggest that H3S10 phosphorylation modulates H3K9
dimethylation and that H3K9me2 redistribution in the absence of H3S10 phosphorylation correlates with activation
of silent genes that lose the H3K9me2 mark and repression
of active genes that acquire the H3K9me2 mark.
DISCUSSION
In this study we have determined the relationship of JIL-1
kinase mediated H3S10 phosphorylation with gene expression and the distribution of the epigenetic H3K9me2 mark.
We show in wild-type salivary gland cells that the H3S10ph
mark is predominantly enriched at active genes whereas the
H3K9me2 mark is largely associated with inactive genes.
Furthermore, our data demonstrate that discrete binding
peaks of JIL-1 are located preferentially to the 5 -end of
genes near the TSS and that these peaks are coincident with
binding peaks for the chromodomain protein, Chromator,
a known binding partner for JIL-1 (40). This distribution of
JIL-1 around the TSS is similar to that obtained by ChIPSeq by Kellner et al. (13) in Kc cells but differs from that
reported by Regnard et al. (10) using ChIP-chip analysis on
custom tiling arrays in S2 cells. These latter workers found
JIL-1 to be more or less equally distributed along the entire
length of genes; however, this result may be a consequence
of using lower affinity polyclonal antibodies combined with
the lower resolution ChIP-chip approach. Interestingly, we
found that although the enrichment profile of H3S10ph was
highly correlated with that of JIL-1, the two profiles were
not identical. The distribution of the H3S10ph mark was
much broader and peak enriched regions were shifted about
600 bp downstream of the TSS. Modeling of the 3D structure of JIL-1 relative to nucleosome structure suggested that
a reason for this lack of overlap is that JIL-1 may have the
capacity to phosphorylate the tail of H3 of one or more nucleosomes some distance away from its actual binding site
depending on the state of higher order nucleosome packaging.
Comparison of global transcription profiles in salivary
glands from wild-type and JIL-1 null mutant larvae revealed that of the nearly 15 000 genes analyzed the expression levels of 1539 genes changed at least 2-fold in the mutant. Surprisingly, among these genes almost as many (49%)
increased their expression as were downregulated (51%).
This raised the question whether certain classes of genes
were preferentially up- or downregulated and further analysis suggested that in general active genes became repressed,
whereas hitherto inactive genes became activated in the absence of H3S10 phosphorylation. Furthermore, the results
showed that downregulation of genes in the mutant was correlated with higher levels or acquisition of the H3K9me2
mark whereas upregulation of a gene was correlated with
loss of or diminished H3K9 dimethylation. These findings
directly demonstrate that H3S10 phosphorylation is not required for transcription or gene activation in Drosophila as
proposed by Corces et al. (11–13). This conclusion is further
supported by recent experiments by Wang et al. (9) analyz-
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Figure 5. Downregulation of moderate and high expression genes acquiring the H3K9me2 mark in the JIL-1 null mutant. (A) Box plots of RPKM of active
genes (RPKM ≥ 1) in wild-type that acquire the H3K9me2 mark in the JIL-1 null mutant background. The distribution of gene expression between the two
categories was significantly different (P-value < 0.0001; Wilcoxon Rank Sum Test). (B) ChIP analysis of randomly selected active genes in wild-type that
are downregulated in the JIL-1 null mutant background. Histograms show the relative enrichment of chromatin immunoprecipitated by anti-H3K9me2 or
anti-H3S10ph antibodies from third instar larval salivary glands from wild-type (WT) and JIL-1 null mutant larvae. For each experimental condition the
average relative enrichment normalized to the corresponding control immunoprecipitation with anti-GST or anti-IgG antibody from three independent
experiments with standard deviation is shown. The difference in H3K9me2 levels for all five genes between wild-type and JIL-1 null salivary glands was
statistically significant (P-values < 0.05; two-tailed Student’s t-tests).
Nucleic Acids Research, 2014, Vol. 42, No. 9 5465
ing white gene expression where levels of the H3S10ph mark
can be manipulated and correlated with the resulting levels of the H3K9me2 mark using ChIP assays. In wild-type,
there are moderate levels of the H3S10ph mark and low levels of the epigenetic H3K9me2 mark at the white gene resulting in its normal expression. However, in the wm4 allele heterochromatic factors can spread across the inversion breakpoint (5,6,43,44) leading to high levels of H3K9me2 at the
white gene and silencing of gene expression. Interestingly,
Wang et al. (9) demonstrated that this increase in H3K9me2
level can be prevented by ectopic H3S10 phosphorylation
at the white gene restoring gene expression. In contrast,
in the absence of H3S10 phosphorylation as it occurs in
strong JIL-1 hypomorphic mutant backgrounds, there is a
redistribution of heterochromatic factors to ectopic chromosome sites resulting in reduced levels of these factors at
the pericentric heterochromatin (3). This leads to less heterochromatic spreading and lower levels of H3K9me2 at the
white gene in the wm4 inversion, thus allowing for increased
white gene expression (9). Thus, taken together these results are compatible with a model (Figure 7) where gene expression levels are directly correlated with the levels of the
H3K9me2 mark independent of the state of the H3S10ph
mark, which is not required for either transcription or gene
activation to occur. However, H3S10 phosphorylation functions to indirectly regulate transcription by counteracting
H3K9 dimethylation and gene silencing in a finely tuned
balance (3,5,6,8).
That genes in GO categories for salivary gland specific
pathways were particularly affected by downregulation in a
JIL-1 mutant background suggests that H3S10 phosphorylation may serve to keep genes transcriptionally active
in a tissue and/or developmentally stage-specific context.
How JIL-1 and H3S10 phosphorylation get targeted to specific sets of genes is not known. However, it has been sug-
Figure 7. Model for indirect regulation of gene expression by H3S10 phosphorylation. (A) At inactive genes, high levels of H3K9 dimethylation lead
to a compact chromatin configuration and gene silencing. (B–D) At active
genes, H3S10 phosphorylation counteracts H3K9 dimethylation leading
to a more open chromatin structure facilitating gene expression. In this
model, gene expression levels are directly correlated with the levels of the
H3K9me2 mark independent of the state of the H3S10ph mark, which is
not required for either transcription or gene activation to occur.
gested as a general model that JIL-1 targeting to active chromatin may be facilitated by or dependent on the presence
of the H3K36me2 and H4K16ac marks (10). Interestingly,
the GO categories for genes becoming activated or upregulated in the absence of H3S10 phosphorylation were enriched in DNA repair, DNA and RNA metabolism, and
cellular response to stress pathways. Upregulation of such
genes would be consistent with a cellular attempt to compensate for the general downregulation of active genes and
to the gross perturbation of chromatin structure occurring
in JIL-1 null mutant backgrounds (2,45). If this represents a
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Figure 6. Upregulation of inactive genes losing the H3K9me2 mark in the JIL-1 null mutant. (A) Box plots of RPKM of inactive genes (RPKM <
1) in wild-type that lose the H3K9me2 mark in the JIL-1 null mutant background. The distribution of gene expression between the two categories was
significantly different (P-value < 0.005; Wilcoxon Rank Sum Test). (B) ChIP analysis of randomly selected inactive genes in wild-type that are upregulated
in the JIL-1 null mutant background. Histograms show the relative enrichment of chromatin immunoprecipitated by anti-H3K9me2 or anti-H3S10ph
antibodies from third instar larval salivary glands from wild-type (WT) and JIL-1 null mutant larvae. For each experimental condition the average relative
enrichment normalized to the corresponding control immunoprecipitation with anti-GST or anti-IgG antibody from three independent experiments with
SD is shown. The difference in H3K9me2 levels for all five genes between wild-type and JIL-1 null salivary glands was statistically significant (P-values <
0.05; two-tailed Student’s t-tests).
5466 Nucleic Acids Research, 2014, Vol. 42, No. 9
specific response to alterations in the mutant, it implies activation of unknown gene induction pathways independent of
H3S10 phosphorylation. However, an alternative scenario
is that when the H3S10ph mark, which normally serves to
limit heterochromatic spreading, is lost in the JIL-1 mutant,
the H3K9me2 silencing mark can now disperse away from
previously repressed locations, resulting in transcriptional
activation of the now unmasked genes.
In summary, the findings of this study indicate that a major functional role of JIL-1-mediated H3S10 phosphorylation is to maintain active gene expression by serving as a
protective epigenetic mark counteracting H3K9 dimethylation and gene silencing. This suggests that different gene expression profiles are regulated by strategic deployment of silencing marks within the genome, and that H3S10 phosphorylation can be an effective means of counteracting silencing
effects. This may also be relevant in other organisms where
H3S10ph is implicated in the rapid yet transient induction
of promoters in response to various inducers (46,47).
The ChIP-Seq and RNA-Seq data have been deposited
in the NCBI Gene Expression Omnibus (http://www.ncbi.
nlm.nih.gov/geo) under accession number GSE41047.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online, including Supplementary Figures S1–S4 and Supplementary Table S1.
ACKNOWLEDGMENTS
We thank Dr D. Nettleton and members of the laboratory
for discussion and advice. We also wish to acknowledge Mr
Atrez Norwood and Mr Cheng-Ting Yeh for technical assistance.
FUNDING
National Institutes of Health [GM062916 to K.M.J. and
J.J.].
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